甘蔗几丁质酶基因ScChiⅣ1的克隆、序列分析及其表达  

Cloning, Sequencing and Expression Analysis of Chitinase Gene ScChiⅣ1 from Sugarcane

作　　者：许颖[1,2] 王竹青[1] 吴期滨 陈云[1] 杨春春 冯敏谢 苏亚春[1] 

机构地区：[1]福建农林大学农业部福建甘蔗生物学与遗传育种重点实验室,福州350002 [2]华中农业大学,武汉430070

出　　处：《基因组学与应用生物学》2015年第6期1258-1266,共9页Genomics and Applied Biology

基　　金：福建省自然科学基金项目(2015J05055)资助

摘　　要：几丁质酶(Chitinase,EC 3.2.2.14)参与植物的生长发育及防卫反应。本研究借助电子克隆技术,以甘蔗(Saccharum spp.)抗黑穗病基因型崖城05-179针刺接种黑穗病菌(Sporisorium scitamineum)48 h的蔗芽c DNA为实验材料,克隆获得一个甘蔗几丁质酶c DNA序列(Gen Bank登录号:KP165001),命名为Sc ChiⅣ1。生物信息学分析结果显示,该核苷酸序列全长为1 037 bp,包含1个长为825 bp的完整开放读码框,编码274个氨基酸,理论等电点为8.29。该基因推导的编码蛋白属于糖苷水解酶19家族成员,含信号肽和几丁质结合区,胞外定位的概率为0.820,推测Sc ChiⅣ1是一种胞外分泌蛋白,同时兼具溶菌酶活性。聚类结果显示,Sc ChiⅣ1归属于ClassⅣ几丁质酶基因。荧光定量PCR分析显示,甘蔗接种黑穗病菌后的96 h内,抗、感基因型中Sc ChiⅣ1表达量均高于对照,且在亲和互作中Sc ChiⅣ1转录本上升应答快(6 h),并在接种黑穗病菌后96 h持续更长,其在甘蔗不同黑穗病抗/感基因型材料(崖城05-179/柳城03-182)与黑穗病菌互作中的表达模式存在差异,但总体结果表明Sc ChiⅣ1受黑穗病菌胁迫诱导表达。本研究为后续甘蔗几丁质酶功能鉴定及甘蔗抗黑穗病基因工程研究奠定基础。Chitinase(EC 3.2.2.14) is involved in the growth and defense responses of plant. c DNA from sugarcane(Saccharum spp.) disease-resistant genotype Yacheng05-179 post inoculation with smut pathogen(Sporisorium scitamineum) 48 h was used as cloning sample. One c DNA sequence of sugarcane chitinase gene, named Sc ChiⅣ1(Gene Bank Accession No. KP165001), was cloned based on in silico cloing method. The bioinformatics analysis showed that the length of Sc ChiⅣ1 gene was 1 037 bp, which contained a length of 825 bp complete open reading frame, encoding 274 amino acids and its theoretical isoelectric point was 8.29. The protein encoded by Sc ChiⅣ1gene belonged to glycoside hydrolase family 19, which contained a signal peptide and a chitin binding domain. It was predicted to be an extracellular hydrophilic secreted protein(0.820 probability) and may have lysozyme activity.Clustering results showed that Sc ChiⅣ1 gene was one of a class Ⅳ chitinase family members in sugarcane. The q RT-PCR results indicated that after inoculation with smut 0 h^96 h, the expression of the Sc Chi Ⅳ 1 gene was higher than that in the mock. It showed more positive early(6 h) response and maintained increased(96 h) transcripts in the compatible interaction than that in the incompatible one. Though the gene expression patterns of Sc ChiⅣ1were different in sugarcane resistant(Yacheng05-179) and susceptible(Liucheng03-182) genotypes challenged with S. scitamineum, the analysis showed that the expression of Sc Chi Ⅳ 1 gene was induced by smut pathogen.These results provided the basis for further functional determination of the chitinases and the smut resistance genetic engineering research in sugarcane.

关 键 词：甘蔗 几丁质酶基因 黑穗病菌 生物信息学分析 定量表达 

分 类 号：S566.1[农业科学—作物学]